WO2017159976A1 - Putrescine-producing microorganism and method for producing putrescine using same - Google Patents

Abstract

The present application relates to a putrescine-producing microorganism in which the activity of formate dehydrogenase is activated, and a method for producing putrescine using the same.

Description

Microorganisms that produce putrescine and methods for producing putrescine using the same

The present application relates to a microorganism producing putrescine and a method of producing putrescine using the same.

Putrescine is known as one of the raw materials of polyamide. Putrescine is produced by chemical methods using petroleum compounds as a raw material, and putrescine fermentation production technology using genetic engineering technology and fermentation technology has been studied.

For example, a strain capable of producing putrescine by manipulating the metabolic pathway of a strain of Corynebacterium is known in Korea (Korean Patent Publication No. 2014-0115244, International Publication WO2014-148743).

On the other hand, formic acid dehydrogenase is an enzyme that catalyzes the oxidation of formic acid to reduce the second substrate, NAD ⁺ , and to produce NADH and CO ₂ as a result. NADH is known to be an important substance throughout the metabolism of strains. This is because it is possible to increase the reducing power in the strain through the increase of NADH may be advantageous to the production of the target material.

It is known to formic NADH using formic acid dehydrogenase to produce succinic acid and bioalcohol under anaerobic conditions. Succinic acid can be produced using the reverse TCA route under anaerobic fermentation conditions. The amount of NADH in the reducing TCA pathway is directly related to succinic acid production, and 2 moles of NADH are consumed from the oxaloacetate to the succinic acid pathway. Indeed, the production of succinic acid from glucose in anaerobic conditions has been reported to increase the yield of more than 20% (Appl Environ Microbiol., 2012, 78 (9): 3325-3337). However, unlike succinic acid, NADH is not used as a direct substrate in the putrescine biosynthetic pathway, and no association has been reported between formic acid dehydrogenase and putrescine production.

The present inventors have made an effort to increase the production of putrescine in a putrescine producing strain, resulting in overexpression of formic acid dehydrogenase to increase the level of NADH and ATP in the strain, thereby increasing the putrescine production. Was completed.

One object of the present application is to provide a microorganism of the genus Corynebacterium producing putrescine with increased activity of formate dehydrogenase (Fdh).

Another object of the present invention is to provide a method for producing putrescine using the microorganism.

The microorganism of the genus Corynebacterium with improved putrescine production capacity is modified to increase the activity of formic acid dehydrogenase, thereby increasing NADH and ATP production, thereby increasing the production of putrescine, thereby increasing the mass production of putrescine. Can be effectively used.

1 is an SDS-PAGE gel photograph showing the results of overexpressing CbFdh using E. coli host. Column 1 is the result of protein in cell pulverized solution expressed for 24 hours at 18 ℃ using E. coli BL21 DE3. Row 2 is the result of soluble protein expressed for 24 hours at 18 ℃ using E. coli BL21 DE3. Row 3 is the result of protein in cell pulverized solution expressed for 8 hours at 30 ° C. using E. coli BL21 DE3. Row 4 is the result of soluble protein expressed for 8 hours at 30 ℃ using E. coli BL21 DE3. Row 5 is the result of the protein of the cell grinding liquid expressed by E. coli Rosetta DE3 for 24 hours at 18 ℃ conditions. Row 6 is the result of soluble protein expressed for 24 hours at 18 ℃ using E. coli Rosetta DE3. Column 7 is the result of protein in the cell pulverized solution expressed for 8 hours at 30 ℃ using E. coli Rosetta DE3. Column 8 is the result of soluble protein expressed for 8 hours at 30 ℃ using E. coli Rosetta DE3.

2 is a graph showing the amount of NADH produced over time. Buffer was used 100 mM phosphate buffer (pH 7.2), the control is the reaction sample excluding soluble protein. CbFdh is a sample of 10% addition of soluble protein overexpressing formic acid dehydrogenase at 30 ° C. using E. coli BL21 DE3. In the case of CbFdh, it was confirmed that NADH, which is a reactant of CbFdh, continuously increased over time.

3 is a graph showing formic acid concentration over time. The control group is a strain in which the pSCEC_CJ7 vector is inserted into a Corynebacterium glutamicum strain. CbFdh is a strain into which the pSCEC_CJ7_CbFdh plasmid capable of expressing the C. boidinii- derived formic acid dehydrogenase gene is inserted. 0, 2, and 10 g / l formic acid were added to the culture, and the change in formic acid concentration of the control group and CbFdh was observed.

As one aspect for achieving the above object, the present application provides a microorganism of the genus Corynebacterium for producing putrescine, the activity of formate dehydrogenase (Fdh) is increased.

As used herein, the term “formate dehydrogenase (hereinafter referred to as“ Fdh ”)” refers to enzymes that form formic acid as a substrate to catalyze oxidation reactions to reduce NAD ⁺ and produce NADH and CO ₂ .

The formic acid dehydrogenase is not limited to its origin or sequence because a difference may exist in the amino acid sequence of a protein exhibiting activity depending on the species or strain of the microorganism.

Specifically, Fdh is a serifoliopsis sub vermispora ( Ceriporiopsis) subvermispora), a bacterium methyl Solarium X Torr quenched switch (Methylobacterium extorquens ), Methylosinus tricosporium trichosporium), the queue-free father Douce oxalato tea kusu (Cupriavidus oxalaticus ), Candida methylica , methylotrophic bacterium , Ancylobacter aquaticus , Komagataella pastoris pastoris ), Mycobacterium vaccae , Arabidopsis thaliana , etc., recently known Corynebacterium glutamicum (Microbiology (2012), 158, 2428-2439) May be derived. Specifically, it may be derived from Candida boidinii , but is not limited thereto.

In addition, Fdh herein is a protein comprising the amino acid sequence of SEQ ID NO: 10, or 70% or more, specifically 80% or more, more specifically 90% or more, even more specifically 95% or more of the sequence and the sequence Specifically, as the amino acid sequence showing a homology of 99% or more, any protein substantially having activity as formic acid dehydrogenase may be included without limitation.

As long as the sequence is homologous to the sequence and has an amino acid sequence having a biological activity substantially the same as or corresponding to that of the protein of SEQ ID NO: 10, the case of some sequences having an amino acid sequence deleted, modified, substituted or added is also the scope of the present application. Inclusion in is self-evident.

The polynucleotide encoding the Fdh herein has at least 70%, in particular at least 80%, more specifically at least 90%, even more than the amino acid sequence of SEQ ID NO: 10 or the sequence as long as it has similar activity as the Fdh protein. Specifically, it may include a polynucleotide encoding a protein exhibiting homology of at least 95%, most specifically at least 99%. For example, it may include the nucleotide sequence of SEQ ID NO: 9.

In addition, the polynucleotide encoding the Fdh herein may be hybridized under stringent conditions with a nucleotide sequence of SEQ ID NO: 9 or a probe derived from the nucleotide sequence, and may be a variant that encodes a normally functioning Fdh. Can be.

As used herein, the term “homology” means a degree of agreement with a given amino acid sequence or base sequence and may be expressed as a percentage. In this specification, homologous sequences thereof having the same or similar activity as a given amino acid sequence or base sequence are designated as "% homology". For example, using standard software that calculates parameters such as score, identity and similarity, in particular BLAST 2.0, or by hybridization experiments used under defined stringent conditions Appropriate hybridization conditions, which are defined within the scope of the art, are well known to those skilled in the art, and are well known to those skilled in the art (e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, New York, 1989; FM Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., New York. As used herein, the term "stringent conditions" refers to conditions that enable specific hybridization between polynucleotides. For example, such conditions are described specifically in the literature (eg, J. Sambrook et al., Homology).

As used herein, the term "increase in activity" means that the activity is improved compared to the intrinsic activity or the activity before the modification of the microorganism. The increase in activity may include both introducing foreign Fdhs and enhancing intrinsic Fdh activity. Specifically, it may mean that the activity of formic acid dehydrogenase is increased and putrescine production capacity is increased.

Specifically, the increase in activity herein

1) an increase in the number of copies of the polynucleotide encoding the enzyme,

2) modification of expression control sequences to increase expression of the polynucleotide,

3) modification of the polynucleotide sequence on the chromosome to enhance the activity of the enzyme, or

4) but may be performed by a method such as deformation to be enhanced by a combination thereof, but is not limited thereto.

The increase in the number of copies of the polynucleotide 1) is not particularly limited thereto, but may be performed in a form operably linked to a vector or by insertion into a chromosome in a host cell. Specifically, a vector capable of replicating and functioning independently of the host, to which the polynucleotide encoding the enzyme of the present application is operably linked, can be performed by introducing into a host cell. Or a vector capable of inserting the polynucleotide into a chromosome in a host cell, to which the polynucleotide is operably linked, is introduced into the host cell to increase the copy number of the polynucleotide in the chromosome of the host cell. have.

In addition, as an aspect of increasing the copy number, it may be carried out by introducing a foreign polynucleotide exhibiting the activity of the enzyme or a codon optimized variant polynucleotide of the polynucleotide. Introduction of the foreign polynucleotide sequence may be performed by introducing into the host cell a foreign polynucleotide encoding an enzyme that exhibits the same / similar activity as the enzyme. The foreign polynucleotide can be used without limitation in its origin or sequence as long as it exhibits the same / similar activity as the enzyme. In addition, the foreign polynucleotide may be introduced into the host cell by optimizing its codons to achieve optimized transcription and translation in the host cell. The introduction can be carried out by a person skilled in the art to appropriately select a known transformation method, the expression of the introduced polynucleotide in the host cell can be produced by the enzyme can be increased its activity.

Next, 2) modification of the expression control sequence to increase the expression of the polynucleotide is not particularly limited, but deletion, insertion, non-conservative or conservative substitution of these nucleic acid sequences to further enhance the activity of the expression control sequence or these It can be carried out by inducing a variation in the sequence in combination with or by replacing with a nucleic acid sequence having a stronger activity. The expression control sequence may include, but is not particularly limited to, a promoter, an operator sequence, a sequence encoding a ribosomal binding site, a sequence that controls the termination of transcription and translation, and the like.

Specifically, a strong heterologous promoter may be linked to the top of the polynucleotide expression unit, but examples of the strong promoter include a CJ7 promoter, a lysCP1 promoter, an EF-Tu promoter, a groEL promoter, an aceA or aceB promoter, and the like. More specifically, the expression rate of the polynucleotide encoding the enzyme is operably linked to the lysCP1 promoter (WO2009 / 096689) or the CJ7 promoter (Korean Patent No. 0620092 and WO2006 / 065095), which are promoters of the genus Corynebacterium. It can be improved, but is not limited thereto.

In addition, 3) modification of the polynucleotide sequence on the chromosome is not particularly limited, and expression control sequences by deletion, insertion, non-conservative or conservative substitution, or a combination thereof, to further enhance the activity of the polynucleotide sequence. This can be done by inducing a phase shift or by replacing with a polynucleotide sequence that has been modified to have stronger activity.

Finally, 4) modification to enhance by the combination of 1) to 3), the increase in the number of copies of the polynucleotide encoding the enzyme, the modification of the expression control sequence to increase its expression, the polynucleotide on the chromosome One or more methods of modifying the sequence and modifying a foreign polynucleotide or a codon-optimized variant polynucleotide thereof that exhibit the activity of the enzyme can be performed together.

As used herein, the term "vector" refers to a DNA preparation containing a nucleotide sequence of a polynucleotide encoding said target protein operably linked to a suitable regulatory sequence to enable expression of the target protein in a suitable host. The regulatory sequence may comprise a promoter capable of initiating transcription, any operator sequence for regulating such transcription, a sequence encoding a suitable mRNA ribosomal binding site, and a sequence regulating termination of transcription and translation. After being transformed into a suitable host cell, the vector can be replicated or function independent of the host genome and integrated into the genome itself.

The vector used herein is not particularly limited as long as it can replicate in a host cell, and any vector known in the art may be used. Examples of commonly used vectors include natural or recombinant plasmids, cosmids, viruses and bacteriophages. For example, pWE15, M13, MBL3, MBL4, IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc. can be used as a phage vector or cosmid vector, and pBR-based, pUC-based, pBluescriptII-based, etc. , pGEM-based, pTZ-based, pCL-based and pET-based and the like can be used. Specifically, pDZ, pACYC177, pACYC184, pCL, pECCG117, pUC19, pBR322, pMW118, pCC1BAC vector and the like can be used.

For example, a polynucleotide encoding a target protein in a chromosome can be replaced with a mutated polynucleotide through an intracellular chromosome insertion vector. Insertion of the polynucleotide into the chromosome can be made by any method known in the art, such as, but not limited to, homologous recombination.

The term "transformation" herein means introducing a vector comprising a polynucleotide encoding a target protein into a host cell so that the protein encoded by the polynucleotide can be expressed in the host cell. The transformed polynucleotides may include all of them, as long as they can be expressed in the host cell, either inserted into the chromosome of the host cell or located outside the chromosome. The polynucleotide also includes DNA and RNA encoding the target protein. The polynucleotide may be introduced in any form as long as it can be expressed by being introduced into a host cell. For example, the polynucleotide may be introduced into a host cell in the form of an expression cassette, which is a gene construct containing all elements necessary for self expression. The expression cassette may include a promoter, a transcription termination signal, a ribosomal binding site, and a translation termination signal, which are typically operably linked to the polynucleotide. The expression cassette may be in the form of an expression vector capable of self replication. In addition, the polynucleotide may be introduced into the host cell in its own form and operably linked with a sequence required for expression in the host cell, but is not limited thereto.

In addition, the term "operably linked" means that the gene sequence is functionally linked with a promoter sequence for initiating and mediating the transcription of a polynucleotide encoding a protein of interest herein.

As used herein, the term "microorganisms that produce putrescine" or "microorganisms having putrescine-producing ability" means that microorganisms having putrescine-producing ability or putrescine in a parent strain lacking the production capacity of putrescine are known. Means a microorganism given production capacity.

The microorganism producing the putrescine is not particularly limited thereto, for example, acetylglutamate synthase or acetylornithine, which converts glutamate to acetylglutamate to enhance the biosynthetic pathway from glutamate to ornithine. Ornithine acetyltransferase (ArgJ), which converts to ornithine, acetylglutamate kinase (ArgB), which converts acetylglutamate to acetylglutamyl phosphate, and acetylglutamate semialdehyde (N acetyl gamma glutamyl phosphate reductase (ArgC) to convert to -acetylglutamate semialdehyde) and acetylornithine aminotransferase (ArgD) to convert acetylglutamate semialdehyde into acetylornithine (N-acetylornithine) Gender is deformed so as to increase than this may be increased productivity of ornithine is used as a raw material gods putrescine biosynthesis.

In addition, the microorganism may be ornithine carbamoyltransfrase (ArgF), a protein that exhibits glutamate exporter activity, and / or an acetyltransfer that acetylates putrescine, which is involved in arginine synthesis in ornithine. The activity of the lagase may be modified to inactivate the intrinsic activity and / or modified to introduce the activity of ornithine decarboxylase (ODC).

At this time, the ornithine carbamoyl transferase (ArgF), the protein showing glutamate exporter activity, ornithine decarboxylase (ODC), ornithine acetyltransferase (ArgJ), acetylglutamate Kinases (ArgB), acetyl gamma glutamyl phosphate reductase (ArgC), and acetylornithine aminotransferase (ArgD) are not particularly limited, but are specifically SEQ ID NOs: 11, 12, 13, 14, respectively. , At least 70%, specifically at least 80%, more specifically at least 90%, even more specifically at least 95%, most specifically at least 99% homology It may include an amino acid sequence having a.

In addition, the acetyltransferase for acetylating putrescine is not particularly limited, but specifically the amino acid sequence represented by SEQ ID NO: 18 or 19 or 70% or more, specifically 80% or more, more specifically Amino acid sequences having at least 90%, more specifically at least 95% and most specifically at least 99% homology.

In addition, the activity of the protein exhibiting putrescine releasing activity may be increased compared to the intrinsic activity, but is not limited thereto. A protein exhibiting putrescine excretion activity is not particularly limited, but specifically, an amino acid sequence of SEQ ID NO: 20 or 21 or 70% or more thereof, specifically 80% or more, more specifically 90% or more And even more specifically at least 95%, most specifically at least 99% homology.

Meanwhile, the microorganism of the present application is a microorganism having a putrescine-producing ability, and may include a prokaryotic microorganism in which the Fdh protein is expressed, and examples thereof include Escherichia sp. And Shigella sp. ), Citrobacter sp., Salmonella sp., Enterobacter sp. Yersinia sp., Klebsiella sp. ( Erwinia sp.), Corynebacterium sp., Brevibacterium sp., Lactobacillus sp., Selenomanas sp., Vibrio genus ( Vibrio sp.), Pseudomonas sp., Streptomyces sp., Arcanobacterium sp., Alcaligenes sp. Specifically, the microorganism of the present application may be a microorganism belonging to the genus Corynebacterium or Escherichia, more specifically Corynebacterium glutamicum ( Corynebacterium glutamicum ), but is not limited thereto.

In another aspect, the present application provides the use of the Corynebacterium microorganism for producing putrescine. The microorganism is a microorganism in which the activity of formate dehydrogenase (Fdh) is increased compared to the activity before modification, and the use may be for producing putrescine.

As another embodiment, the present invention comprises the steps of (a) culturing in the medium a microorganism of the genus Corynebacterium producing putrescine with increased activity of formate dehydrogenase (Fdh); And (b) recovering putrescine from the microorganism or medium obtained in the step.

Microorganisms having improved formic acid dehydrogenase and putrescine production capacity have been described above.

In the above method, the step of culturing the microorganism is not particularly limited thereto, and may be performed by a known batch culture method, continuous culture method, fed-batch culture method, or the like. At this time, the culture conditions are not particularly limited thereto, but using a basic compound (eg, sodium hydroxide, potassium hydroxide or ammonia) or an acidic compound (eg, phosphoric acid or sulfuric acid), an appropriate pH (eg, pH 5 to 9, specifically, Can adjust pH 6 to 8, most specifically pH 6.8), and maintain an aerobic condition by introducing oxygen or oxygen-containing gas mixture into the culture. The culture temperature may be maintained at 20 to 45 ℃, specifically 25 to 40 ℃, can be incubated for about 10 to 160 hours, but is not limited thereto. Putrescine produced by the culture may be secreted into the medium or remain in the cells.

In addition, the culture medium used includes sugars and carbohydrates (e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose), fats and fats (e.g. soybean oil, sunflower seeds) as carbon sources. Oils, peanut oils and coconut oils), fatty acids (e.g. palmitic acid, stearic acid and linoleic acid), alcohols (e.g. glycerol and ethanol) and organic acids (e.g. acetic acid) may be used individually or in combination. This is not restrictive. Nitrogen sources include nitrogen-containing organic compounds (eg peptone, yeast extract, gravy, malt extract, corn steep liquor, soybean meal and urea), or inorganic compounds (eg ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and Ammonium nitrate) and the like can be used individually or in combination, but is not limited thereto. As a source of phosphorus, potassium dihydrogen phosphate, dipotassium hydrogen phosphate, a corresponding sodium-containing salt, and the like may be used individually or in combination, but is not limited thereto. The medium may also contain essential growth-promoting substances such as other metal salts (eg magnesium sulfate or iron sulfate), amino acids and vitamins.

The method of recovering putrescine produced in the culturing step of the present application may collect the desired amino acid from the culture medium using a suitable method known in the art according to the culture method. For example, centrifugation, filtration, anion exchange chromatography, crystallization and HPLC can be used, and the desired putrescine can be recovered from the medium or microorganism using any suitable method known in the art.

Hereinafter, the present application will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only, and the scope of the present application is not limited by these examples.

Example 1: E. coli expression and reactivity evaluation of CbFdh

1) E. coli expression of CbFdh gene

CbFdh ( Candida To overexpress boidinii formate dehydrogenase in E. coli Candida boidinii ) KCTC17776 strain was cultured to obtain genomic DNA. The formic acid dehydrogenase gene (CbFdh) (SEQ ID NO: 9) was inserted into the pET28a vector using the primers of SEQ ID NOs: 1 and 2.

Specifically, PCR conditions were repeated 30 times for denaturation 95 ℃ 30 seconds, annealing (annealing) 55 ℃ 30 seconds, extension (72 ℃) 1 minute. After electrophoresis on a 1.0% agarose gel from the PCR result, a 1.1 kb band was eluted and purified. NcoI and XhoI were added to the purified PCR product and the pET28a vector solution sample, followed by restriction enzyme treatment at 37 ° C. for 4 hours. Purified nucleic acid fragments were obtained using kit (GeneAll, Seoul). Each of the obtained 1 mg CbFdh fragment and pET28a vector fragment were ligated using T4 ligase and then electroporated at 2500 V to the E. coli DH5α strain. After the electroporation, the recovered strains were plated in LB plate medium containing 50 μg / L spectinomycin and cultured at 37 ° C. for 1 day, and then strains showing resistance to spectinomycin were selected. The recovered strains were plated in LB plate medium containing 50 μg / L kanamycin and cultured at 37 ° C. for 1 day, and the strains showing resistance were selected. The selected strain was PCR under the above conditions using primers of SEQ ID NOs: 3 and 4 of the T7 promoter and terminator sequence, and then inserted into the CbFdh by confirming that the gene size was observed to be 1.3 kb on 1.0% agarose gel. It was confirmed.

The strain in which the insertion of CbFdh was confirmed was placed in 50 ml / ml of ampicillin in 3 ml of LB solution medium and incubated at 37 ° C. for 12 hours. Cultured strains were placed in 50 ml LB containing antibiotics and cultured at 37 ° C., and the optical luminosity (600 nm wavelength) became 0.8. 0.2 mM IPTG was added to induce expression at various temperature / time conditions. The cultured strains were washed and cells were crushed using a sonicator. After grinding, the SDS-PAGE gel result confirmed that CbFdh (41kDa, SEQ ID NO: 10) was overexpressed (FIG. 1).

2) Evaluation of activity of expressed CbFdh gene

To assess the activity of CbFdh, 100 mM phosphate buffer (pH 7.2) was used as reaction buffer. 10 mM NAD ⁺ and 0.1% sodium formate were added to the buffer solution as a control. On the other hand, the CbFdh overexpressing cell pulverized solution confirmed in Example 1-1) was added to the control group at a 10% rate to evaluate the activity of CbFdh. The reaction solution was checked for numerical changes at 339 nm wavelength using a 96 well plate reader. Light of 340 nm wavelength is known to be selectively absorbed by NADH.

As a result, it was confirmed that NADH is continuously produced for several minutes (Fig. 2). In this example, CbFdh can be overexpressed in E. coli, and the expressed protein was evaluated as having inherent activity.

Example 2: Preparation of microorganisms of the genus CbFdh expressing Corynebacterium

Next, to enhance the function of putrescine production by enhancing the function of CbFdh in the strains of Corynebacterium genus to produce putrescine. In order to express CbFdh in Corynebacterium sp. Strain and confirm its activity, a CJ7 promoter (KCCM10617, Republic of Korea Patent No. 10-0620092) was introduced before the gene start codon of CbFdh.

First, PCR was performed using primer pairs of SEQ ID NOs: 5 and 6 as a template using genomic DNA of Corynebacterium glutamicum ATCC13032 to obtain a gene including the CJ7 promoter sequence. The PCR reaction was performed by repeating the procedure of denaturation 95 ℃ 30 seconds, annealing 55 ℃ 30 seconds and elongation 72 ℃ 30 seconds 30 times. PCR nucleic acid product having a size of 400 bp (base pair) after electrophoresis was confirmed using 1.5% agarose gel. From the obtained PCR result, a purified CJ7 promoter nucleic acid fragment was obtained using a PCR prep kit (GeneAll, Seoul). BamHI and XbaI were added to the purified CJ7 nucleic acid fragment and the pSCEC vector solution sample, followed by restriction enzyme treatment at 37 ° C. for 4 hours. Then, after electrophoresis, a 1.5% agarose gel was used to cut the CJ7 promoter and pSCEC vector-sized nucleic acid fragments having a size of 400 bp. Nucleic acid fragments were obtained. Each 1 mg of CJ7 promoter fragment and pSCEC vector were linked using T4 ligase and then electroporated to 2500 V in E. coli DH5α strain. Strains recovered after electroporation were plated in LB plate medium containing 50 μg / L spectinomycin and cultured at 37 ° C. for 1 day, and 18 strains showing resistance to spectinomycin were selected. 18 selected strains were identified using PCR primers of SEQ ID NOs: 5 and 6, and PCR products having a size of 400 bp after colony PCR. The production of pSCEC_CJ7 having a CJ7 promoter was confirmed from colony PCR results.

The CbFdh PCR product that can be inserted into pSCEC_CJ7 was obtained using the primers of SEQ ID NOs: 7 and 8 in the same manner as the CbFdh PCR product obtaining condition obtained in Example 1 above. PSCEC_CJ7 and CbFdh PCR products treated with restriction enzymes XbaI and SalI were linked to the E. coli DH5α strain. PSCEC_CJ7_CbFdh was obtained from the selected strains and electroporated at 2500 V in the microorganisms of the Corynebacterium genus, the putrescine-producing strain KCCM11240P (Korean Patent No. 2013-0082478) and KCCM11401P (Korean Patent No. 2014-0017243). (electrophoration).

Colonies were cultured by plating the strains obtained by electroporation in BHIS plate medium (Braine heart infusion 37 g / l, sorbitol 91 g / l, agar 2%) containing 50 μg / l spectinomycin. Formed. The selected strains were CM medium containing 50 μg / l spectinomycin (glucose 10 g / l, polypeptone 10 g / l, yeast extract 5 g / l, beef extract 5 g / l, NaCl 2.5 g / 1 L, Urea 2 g / L, pH 6.8) was finally selected by shaking culture. KCCM11240P strain with pSCEC_CJ7_CbFdh inserted was named KCCM11240P / pSCEC_CJ7_CbFdh (CC04-0081) and KCCM11240P strain with pSCEC_CJ7 was named KCCM11240P / pSCEC_CJ7. Similarly, the KCCM11401P strain into which pSCEC_CJ7_CbFdh was inserted was named KCCM11401P / pSCEC_CJ7_CbFdh, and the KCCM11401P strain into which pSCEC_CJ7 was inserted was named KCCM11401P / pSCEC_CJ7.

Among them, the CC04-0081 strain was deposited with the accession number KCCM11798P to the Korea Culture Center of Microorganisms (KCCM), an international depository institution under the Budapest Treaty on January 8, 2016.

Example 3: Evaluation of CbFdh Activity in Microorganisms of the Genus Corynebacterium

In order to confirm formic acid decarboxylase activity of the strain of Corynebacterium sp. Into which CbFdh was inserted, formic acid concentration change was analyzed in a medium containing formic acid (FIG. 3). Formic acid at concentrations of 0 g / L, 2 g / L, and 10 g / L was added to the culture medium of the CbFdh-enhanced strain and the empty vector-inserted strain. Reinforcing strains and empty vector insertion strains were KCCM11240P / pSCEC_CJ7_CbFdh and KCCM11240P / pSCEC_CJ7.

In the case of the strain containing the empty vector as a result of the culture, it was confirmed that formic acid remained in the culture solution when 2 g / L and 10 g / L formic acid was added. On the other hand, the CbFdh-enhanced strains could be found to degrade all of 2 g / L formic acid within 24 hours. In addition, when 10 g / L formic acid was added, all decomposition could not be performed within 32 hours, but it was confirmed that formic acid was continuously decreased. As compared with the control strain at 32 hours after the reaction, it was confirmed that about 80% of formic acid was converted.

Analysis of formic acid change through the culture of CbFdh enriched strain and control strain confirmed that the CbFdh enriched strain degraded formic acid. Through this result, it was confirmed that CbFdh introduced into the strain of Corynebacterium is normally expressed and maintains its function.

Example 4 Evaluation of Production Capacity of CbFdh Enhanced Putrescine Strains

Four kinds of Corynebacterium glutamicum mutants (KCCM11240P / pSCEC_CJ7_CbFdh, KCCM11240P / pSCEC_CJ7, KCCM11401P / pSCEC_CJ7_CbFdh / KCCM1141PJJ) containing KCCM11401PJ / CCEC11401PJJ, respectively containing Flat media (glucose 1%, polypeptone 1%, yeast extract 0.5%, beef extract 0.5%, NaCl 0.25%, urea 0.2%, 50% NaOH 100 μl, 50 μg spectinomycin, agar 2%, pH 6.8, 1 L Base plate) and incubated for 24 hours at 30 ℃. Each strain was incubated with 25 ml of titer medium (Glucose 8%, soy protein 0.25%, corn solid 0.50%, (NH ₄ ) ₂ SO ₄ 4%, KH ₂ PO ₄ 0.1%, MgSO ₄ · 7H ₂ O 0.05%, urea 0.15%, biotin 100 μg, thiamine hydrochloride 3 mg, calcium-pantothenic acid 3 mg, nicotinamide 3 mg, CaCO ₃ 5%, 50 μg spectinomycin, based on 1 L) It was incubated at 200 rpm for 30 hours for 98 hours for the KCCM11240P / pSCEC_CJ7_CbFdh and KCCM11240P / pSCEC_CJ7 strains, and 104 hours for the KCCM11401P / pSCEC_CJ7_CbFdh and KCCM11401P / pSCEC_CJ7 strains.

The putrescine concentration produced from each culture was measured and the results are shown in Table 1 below.

Strain name	Formic acid addition amount (g / ℓ)	Putrescine (g / ℓ)
KCCM11240P / pSCEC_CJ7	0	12.2
KCCM11240P / pSCEC_CJ7	5	12.3
KCCM11240P / pSCEC_CJ7_CbFdh	0	13.4
KCCM11240P / pSCEC_CJ7_CbFdh	5	13.1
KCCM11401P / pSCEC_CJ7	0	11.4
KCCM11401P / pSCEC_CJ7	5	10.7
KCCM11401P / pSCEC_CJ7_CbFdh	0	12.0
KCCM11401P / pSCEC_CJ7_CbFdh	5	12.0

Putrescine concentration present in the culture was analyzed using HPLC. As shown in Table 1, in the case of KCCM11240P / pSCEC_CJ7 strain, putrescine production was not significantly changed depending on the presence of 5 g / L formic acid. On the other hand, the strain introduced KCCM11240P / pSCEC_CJ7_CbFdh was confirmed to increase the production more than 7% compared to the KCCM11240P / pSCEC_CJ7 strain regardless of the presence or absence of 5g / ℓ formic acid. It was confirmed that putrescine production of the Fdh-enhanced strain was increased with or without formic acid.

In addition, 11.4 g / L of putrescine was produced for the KCCM11401P / pSCEC_CJ7 strain evaluated in the same medium without addition of formic acid, and the strains cultured in the medium to which 5 g / L of formic acid was added reduced the yield by about 6% (10.7). g / l). On the other hand, the CbFdh-enhanced strain KCCM11401P / pSCEC_CJ7_CbFdh was confirmed to produce the same amount of putrescine (12.0 g / L) regardless of the presence or absence of formic acid. When analyzing putrescine produced from KCCM11401P / pSCEC_CJ7_CbFdh and KCCM11401P / pSCEC_CJ7 strains, it was confirmed that the CbFdh-enhanced strains increased putrescine production by 5% or more than KCCM11401P / pSCEC_CJ7. With or without formic acid, it was confirmed that the CbFdh-enhanced strain improved putrescine production capacity.

In summary, the transformed strain in which formic acid dehydrogenase (CbFdh) was introduced into the putrescine producing strain was confirmed to further increase the putrescine production, which is an effect regardless of the addition of formic acid. Accordingly, the present invention is expected to be able to efficiently mass-produce putrescine.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, it should be understood that the embodiments described above are exemplary in all respects and not limiting. The scope of the present application should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present application.

Claims (10)

1. A microorganism of the genus Corynebacterium producing putrescine, in which the activity of formate dehydrogenase (Fdh) is increased compared to the activity before transformation.
2. 2. The method of claim 1, wherein the Candida Fdh show dini (Candida boidinii ) from microorganisms.
3. The microorganism of claim 1, wherein the Fdh consists of the amino acid sequence of SEQ ID NO: 10.
4. The microorganism according to claim 1, wherein the activity of ornithine decarboxylase (ODC) is further introduced.
5. The microorganism of claim 1, further wherein the activity of acetyltransferase is weakened relative to intrinsic activity.
6. The microorganism of claim 1, wherein the protein activity exhibiting putrescine excretion activity is increased compared to endogenous activity.
7. The microorganism of claim 1, wherein the microorganism is Corynebacterium glutamicum .
8. (a) culturing the microorganism of any one of claims 1 to 7 in a medium; And

   (b) recovering putrescine from the microorganism or medium obtained in the step, putrescine production method.
9. The method of claim 8, wherein the culturing is culturing the microorganism in a medium free of formic acid.
10. The method of claim 8, wherein culturing the microorganism is culturing in aerobic conditions.

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